L1 and epithelial cell adhesion molecules ... - Semantic Scholar

Wang et al. Journal of Experimental & Clinical Cancer Research 2013, 32:66 http://www.jeccr.com/content/32/1/66

RESEARCH

Open Access

L1 and epithelial cell adhesion molecules associated with gastric cancer progression and prognosis in examination of specimens from 601 patients Yuan-Yu Wang1*, Li Li2, Zhong-Sheng Zhao3*, Yong-Xiang Wang1, Zai-Yuan Ye1 and Hou-Quan Tao2

Abstract Background: L1 cell adhesion molecule (L1CAM) and epithelial cell adhesion molecule (EPCAM) have been implicated in the development and progression of gastric cancer. The present study investigated the clinical significance of L1CAM and EPCAM in the development, progression and prognosis of gastric cancer. Methods: Expression of L1CAM and EPCAM were examined immunochemically in 601 clinicopathologically characterized gastric cancer cases. Results: L1CAM protein was detected in 23.9% of human non-tumor mucosa samples. All samples expressed L1CAM protein at low levels. High expression of L1CAM protein was detected in 163 (27.1%) tumors. Expression of L1CAM correlated with age, tumor location, size of tumors, Lauren’s classification, depth of invasion, lymph node and distant metastases, regional lymph node stage, Tumor-Node-Metastasis (TNM) stage and prognosis. EPCAM protein was detected in 45.7% of human non-tumor mucosa samples. All samples expressed EPCAM protein at low levels. High expression of EPCAM protein was detected in 247 (41.1%) tumors. Expression of EPCAM correlated with age, tumor location, size of tumors, Lauren’s classification, depth of invasion, lymph node and distant metastases, regional lymph node stage, TNM stage and prognosis. Cumulative 5-year survival rates of patients with high expression of both L1CAM and EPCAM were significantly lower than in patients with low expression of both. Conclusions: Expression of L1CAM and EPCAM in gastric cancer was significantly associated with lymph node and distant metastasis, and poor prognosis. L1CAM and EPCAM proteins could be useful markers to predict tumor progression and prognosis. Keyword: Gastric carcinoma, L1CAM, EPCAM, Immunohistochemistry, Progression, Prognosis

Introduction Although global incidence of gastric cancer has decreased in recent years, its mortality rate in China is the highest among all tumors. The main cause of death is invasion and metastasis of tumor. Tumor invasion and metastasis is a very complicated and continuous process involving multiple steps, regulated at the molecular level by * Correspondence: [email protected]; [email protected] 1 Department of Gastrointestinal Surgery, Zhejiang Provincial People’s Hospital, Hangzhou 310014, PR China 3 Department of Pathology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, PR China Full list of author information is available at the end of the article

adhesion molecules, protein catabolic enzymes, cellular growth factors and various angiogenic factors. L1 cell adhesion molecule (L1CAM) is a cell adhesion molecule of the immunoglobulin superfamily of cell adhesion molecules (IgCAM), initially identified in the nervous system. Recent studies demonstrated L1CAM expression in various types of cancer, predominantly at the invasive front of tumors and metastases. Overexpression of L1CAM in normal and cancer cells increased motility, enhanced growth rate and promoted cell transformation and tumorigenicity. The epithelial cell adhesion molecule (EPCAM) is a glycoprotein of approximately 40 kD that was originally identified as a marker for carcinoma. EPCAM’s effects are not

© 2013 Wang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


limited to cell adhesion; they include diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EPCAM may actually prevent cell–cell adhesion. The current study examined expression of L1CAM and EPCAM in surgical specimens of gastric carcinoma, to explore possible correlations between L1CAM and EPCAM expression and clinicopathological variables and prognosis.

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December 2008. Survival times were counted from the dates of surgery to the follow-up deadline or dates of death, which were mostly caused by carcinoma recurrence or metastasis. Ninety-two noncancerous human gastric tissues were obtained from gastrectomies of adjacent gastric cancers beyond margins >5 cm. Routine chemotherapy was given to patients with advanced-stage disease after operation, but no radiation treatment was administered to any patients included in our study.

Materials and methods Cell culture and frozen tissues

Real-time quantitative RT-PCR

Human gastric cancer cell lines AGS, MKN-28, BGC-823, HCG-27, SGC-7901, 9811P, MKN-45 and non-malignant gastric epithelial cell line GES-1 were obtained from Key Laboratory of Gastroenterology of Zhejiang Province (Hangzhou, China), and cultured in RPMI1640 containing 10% foetal bovine serum (FBS), 50U/ml penicillin and 50 μg/ml streptomycin. All cells were maintained at 37°C under an atmosphere of 5% CO2.

Expressions of L1CAM and EPCAM in 42 tumor tissue samples and matched normal gastric mucosa were confirmed by RT-PCR. Total RNA was extracted by TRIzol and cDNAs were reverse-transcribed by RevertAid TM reverse transcriptase. Real-time PCR was carried out using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) at 50°C for 2 min, 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, and at 60°C for 1 min. The primers for GAPDH (224 bp) were 5′-TGAAGGTCGGA GTCAACGG-3′ (sense) and 5′- CTGGAAGATGGTGAT GGGATT-3′ (antisense). The primers for L1CAM (187 bp) were 5′-TGTCCTTCCCTTTACGCCAC-3′ (sense) and 5′- GACCAAGCACAGGCATACAGG-3′ (antisense). The primers for EPCAM (101 bp) were 5′-ATAATAATCGTC AATGCCAGTG-3′ (sense) and 5′- ATTCATTTCTGCCT TCATCAC-3′ (antisense). The expression of GAPDH was used to normalize that of the target genes. Each assay was done in triplicate and the average calculated. The expression level of L1CAM/EPCAM was expressed as 2–ΔΔCt, ΔCt = Ct(Target) – Ct(GAPDH).

Patients and frozen tissue samples

Our study included 42 patients (29 male, 13 female; mean age: 59 years; range: 30–86) collected from gastrectomy specimens from the Department of Surgery, Zhejiang Provincial People’s Hospital from January 2010 and January 2011. None of the patients were treated with radiotherapy or perioperative chemotherapy, and all had undergone total gastrectomies. Resected specimens were studied pathologically according to the criteria described in the AJCC classification (2009). There were 24 tubular adenocarcinomas, 3 papillary adenocarcinomas, 10 mucinous adenocarcinomas, 5 signet-ring cell carcinomas. Two cases were categorized as stage I, 8 as stage II, 29 as stage III, and 3 as stage IV. The study items included age, sex, tumor location, tumor size, gross (Borrmann) type, gastric wall invasion, resection margin, histological type, lymph node metastasis, vascular invasion, lymphatic invasion, and perineural invasion. Fresh samples of tumor tissue, and matched normal gastric mucosa were obtained immediately after gastric resection. The samples were dissected carefully from resected specimens by a pathologist, and immediately snap-frozen in separate vials using liquid nitrogen. These frozen specimens were stored at −80°C in a tumor bank before use. Patients and paraffin-embedded tissue samples

Gastric cancer tissues were collected from gastrectomy specimens of 601 patients from the Department of Surgery, Zhejiang Provincial People’s Hospital from January 1998 to January 2004. Tissues had been formalin-fixed and paraffin-embedded, and clinically and histopathologically diagnosed at the Departments of Gastrointestinal Surgery and Pathology. All patients had follow-up records over at least 5 years. The follow-up deadline was

Tissue microarray

Blocks containing a total of 693 cases (601 cancer samples and 92 non-cancer tissue samples) were prepared as described previously [1,2]. Immunohistochemistry

Immunohistochemical analysis was used to study altered protein expression in 92 noncancerous human gastric tissue controls and 601 human gastric cancer tissues [3,4]. In brief, slides were baked at 60°C for 2 h, followed by deparaffinization with xylene and rehydration. The sections were submerged into EDTA antigenic retrieval buffer and microwaved for antigenic retrieval, after which they were treated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity, followed by incubation with 1% bovine serum albumin to block nonspecific binding. Sections were incubated with rabbit anti-EPCAM(Epitomics), and with mouse anti-L1CAM (Abcam), overnight at 4°C. Normal goat serum was used as a negative control. After washing, tissue sections were treated with secondary antibody. Tissue sections were then counterstained with hematoxylin, dehydrated, and mounted. Cytoplasm with


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Figure 1 Expression of L1CAM mRNA in gastric cancer cell lines.

L1CAM and EPCAM was stained as buffy. The degree of immunostaining was reviewed and scored independently by two observers based on the proportion of positively stained tumor cells and intensity of staining [5-7]. Statistical analysis

All statistical analyses were performed using SPSS16.0 software. Measurement data were analyzed using Student’s t test, while categorical data were studied using χ2 or Fisher exact tests. Survival curves were estimated using the Kaplan–Meier method; the log-rank test was used to compute differences between curves. Multivariate analysis using the Cox proportional hazards regression model was performed to assess prognostic values of protein expression. Correlation coefficients between protein expression and clinicopathological findings were estimated using the Pearson correlation method. Statistical significance was set at P < 0.05.

Results Expression of L1CAM and EPCAM mRNA in gastric tumor tissue and cell lines

L1CAM and EPCAM mRNA were significantly upregulated in AGS, MKN-28, BGC-823, HCG-27, SGC-

Figure 2 Expression of EPCAM mRNA in gastric cancer cell lines.

7901, 9811P and MKN-45 cell lines compared with the non-malignant gastric epithelial cell line GES-1 (P < 0.05, Figure 1, Figure 2). In 42 gastric tumor tissue samples and matched normal gastric mucosa, average expressions of L1CAM were 0.0403 ± 0.0069 and 0.0093 ± 0.0010, respectively, and were significant different (t = 2.845, P = 0.006). L1CAM was over-expressed in 25 gastric tumor tissue samples compared with matched normal gastric mucosa. In 42 gastric tumor tissue samples and matched normal gastric mucosa, the average expressions of EPCAM were 0.4199 ± 0.0485 and 0.1759 ± 0.0144, respectively, and were significantly different (t = 3.122, P = 0.002). EPCAM was over-expressed in 27 gastric tumor tissue samples compared with matched normal gastric mucosa.

Expression of L1CAM and EPCAM in archived gastric cancer tissue and non-cancer mucosa

L1CAM protein was detected in 22/92 (23.9%) human non-tumor mucosa samples; all samples expressed L1CAM protein at low levels. High L1CAM protein expression was detected in 163 (27.1%) tumors. L1CAM was localized mainly in the cytoplasm of primary cancer cells (Figure 3).


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Figure 3 Immunohistochemical staining for L1CAM in gastric cancer lesions (601 case) and noncancerous tissues (92 case). A: L1CAM was negative in noncancerous tissues, B: L1CAM was highly expressed in well differentiated adenocarcinoma, C: L1CAM was highly expressed in moderately differentiated adenocarcinoma, D: L1CAM was highly expressed in poorly differentiated adenocarcinoma.

Figure 4 Immunohistochemical staining for EPCAM in gastric cancer lesions (601 case) and noncancerous tissues (92 case). A: EPCAM was negative in noncancerous tissues, B: EPCAM was highly expressed in well differentiated adenocarcinoma, C: EPCAM was highly expressed in moderately differentiated adenocarcinoma, D: EPCAM was highly expressed in poorly differentiated adenocarcinoma.


Table 1 Relationship of L1CAM expression with pathological parameters of tumor Clinical parameters Age(yrs)

High

PN3 t/χ2/r

P

57.86 ± 11.88 61.20 ± 11.85 3.065

0.002

3.386

0.066

13.39

0.001

Gender Male

321 (75.0%)

107 (25.0%)

Female

117 (67.6%)

56 (32.4%)

Location Proximal

54 (64.3%)

30 (35.7%)

Middle

150 (67.3%)

73 (32.7%)

Distal

234 (79.6%)

60 (20.4%)

Size

26.99 0.0001

0.05; Table 1). Expression of EPCAM correlated with age, tumor location, tumor size, Lauren’s classification, depth of invasion, lymph node and distant metastases, regional lymph node stage and TNM stage (P < 0.05). EPCAM expression did not correlate with sex, differentiation, or histological classification (P > 0.05; Table 2). Correlation between L1CAM and EPCAM expression and patient prognosis

As TNM stage, lymph node and distant metastasis are used as prognostic factors for gastric cancer [8], we further analyzed the correlation between L1CAM/EPCAM expression and patient prognosis according to Lauren classification, TNM stage and regional lymph nodes. Kaplan–Meier curves with univariate analyses (log-rank) for patients with low L1CAM expression versus high L1CAM expression tumors according to Lauren classification, showed significant differences (Table 3, Figure 5), as did Kaplan–Meier curves with univariate analyses (logrank) for patients with low L1CAM expression versus high L1CAM expression tumors according to regional lymph nodes. Cumulative 5-year survival rates for patients with low L1CAM were significantly higher than in patients with high L1CAM expression among those in PN0 and PN1 stages (Table 3, Figure 6). Kaplan–Meier curves with univariate analyses (log-rank) for patients with low L1CAM expression versus high L1CAM expression tumors according to TNM stage, showed cumulative 5-year survival rates for patients with low L1CAM were significantly higher than in patients with high


Table 2 Relationship of EPCAM expression with pathological parameters of tumor Clinical parameters

High

56.85 ± 11.4 61.51 ± 12.22

Gender Male

257 (60.0%)

171 (40.0%)

Female

97 (56.1%)

76 (43.9%)

Location Proximal

37 (44.0%)

47 (56.0%)

Middle

130 (58.3%)

93 (41.7%)

Distal

187 (63.6%)

107 (36.4%)

Size